Gel filtration chromatography (also known as size or molecular exclusion chromatography) separates molecules based on their ability to penetrate into the pores or channels in agarose or dextran beads. As a mixture of molecules in a fluid permeate through the beads of gel the volume available for diffusion is determined by their diameter and the size of the channels in the gel beads. The smaller molecules have a greater volume available to them and therefore take longer to pass through the column. There is no interaction between the molecules to be separated and the column matrix and as a consequence this is a gentle technique with no harsh or potentially denaturing conditions necessary for elution. Indeed virtually any buffer system may be used and, as large protein molecules will be separated from small ions and molecules, the technique can be used to transfer the protein from one buffer to another, or to effect a change in salt concentration. The protein will end up in the buffer in which the column has been equilibrated. However, because diffusion is at the center of the separation process, the protein will become more dilute (by as much as a factor of 10), and the capacity (the amount of protein that can be loaded) is low. As a consequence it is a technique normally used in the final stages of a purification protocol when the protein is in a relatively pure state and there is a sufficient difference in molecular size in the remaining components and as such is suited to the separation of (Fab')2 from contaminating peptides arising from the proteolytic digestion (see Notes 31 and 32).
1. The gel filtration matrix is supplied as a suspension and usually contains a preservative such as ethanol. Wash the matrix (see Note 33) with water on a filtration funnel to remove the ethanol and then wash with the eluting buffer (0.05 M sodium phosphate, 0.15 M NaCl, pH 7.4; see Note 34).
2. Transfer the matrix to a suitable container and allow it to settle. Add or remove water to give a volume equivalent to the settled volume plus 25%.
4. Set up the column (see Note 35) vertically, add 5-10 mL of eluting buffer to the column, and allow approx 5 mL of this to flow through to remove air bubbles, then close off the outlet.
5. Attach a packing reservoir to the top of the column of sufficient volume to allow all of the matrix to be added at the one time.
6. Pour the matrix gently into the reservoir, taking care to avoid air bubbles (pour down a glass rod, one end of which is in contact with the inside of the reservoir).
7. Ensure that the operating pressure is less than the maximum indicated by the manufacturer (see Note 36) and allow the liquid to flow through until the level of the packed matrix is stable (see Note 37).
8. Once the column level is stable, remove the reservoir and equilibrate the column by passing through two-column volumes of the eluting buffer.
184.108.40.206. Sample Application and Chromatographic Separation
1. Carefully remove the liquid from the top of the column and apply the sample without disturbing the gel bed (see Note 38).
2. Allow the sample to run in and, taking care not to allow air to enter the gel bed, wash in with approx 1 mL of eluting buffer (see Note 39).
3. Layer 2-3 mL of eluting buffer on to the column, reinstate the eluting buffer delivery line and adjust the pressure head, or pump to give a flow rate of 0.5 mL/min (see Note 40).
4. Collect 1-2 mL fractions and monitor the absorbance at 280 nm.
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